Deep reptilian evolutionary roots of a major avian respiratory adaptation

Vertebral ribs of the anterior thorax in extant birds bear bony prongs called uncinate processes, which improve the mechanical advantage of mm. appendicocostales to move air through the immobile lung and pneumatic air sacs. Among non-avian archosaurs, broad, cartilaginous uncinate processes are present in extant crocodylians, and likely have a ventilatory function. Preserved ossified or calcified uncinate processes are known in several non-avian dinosaurs. However, whether other fossil archosaurs possessed cartilaginous uncinate processes has been unclear. Here, we establish osteological correlates for uncinate attachment to vertebral ribs in extant archosaurs, with which we inferred the presence of uncinate processes in at least 19 fossil archosaur taxa. An ancestral state reconstruction based on the infer distribution suggests that cartilaginous uncinate processes were plesiomorphically present in Dinosauria and arguably in Archosauria, indicating that uncinate processes, and presumably their ventilatory function, have a deep evolutionary history extending back well beyond the origin of birds.

Thank you for the opportunity to review your work. I enjoyed reading your article. It is nice to know that an osteological correlate like the one you proposed is available. I thought your data sampling among dinosaurs was good but I only counted two non-crocodylian pseudosuchians that you sampled. It would be great to see more of the latter as this will make your reconstruction of the Archosaur ancestral condition more robust. As is, I recommend to narrow your scope to dinosaurs and modern crocodylians as I think this would still make an interesting paper suited for this journal. I hope you find my comments to be helpful. Please feel free to reach out if you have any questions.
I look forward to seeing this out.
Best regards,

Michael Pittman The Chinese University of Hong Kong
Reviewer #2 (Remarks to the Author): This is a well-written and rigorous paper that brings significant new data to an interesting topic: breathing mechanisms in modern and fossil archosaurs. The paper focuses on uncinate processesbony or cartilaginous attachments to the ribs that appear to be important in lung ventilation in modern archosaurs -and demonstrates for the first time an osteological correlate of these processes that allows them to be identified in fossil taxa where the processes may not have been ossified. The distribution of this osteological correlate in fossil taxa is then used to argue that uncinate processes are ancestral for Archosauria.
I am fully supportive of publication with minimal revisions. I note only the following minor points. 1) Various taxonomic names are mentioned at various points without explaining them. For the benefit of the reader I would suggest "anhimid birds" rather than "anhimids", "neognath birds" instead of "neognaths" etc.
2) At more than one point it is asserted that uncinate processes cannot be ancestral for saurians because they are absent in squamates (lizards and snakes). But if they are absent in squamates but present in rhynchocephalians, then the ancestral condition for Lepidosauromorpha is ambiguous, as is the ancestral condition for Sauria. You should tweak your text to reflect this ambiguity.
3) On line 69 thescelosaurids and ornithopods are listed as if they are different taxa. But thescelosaurids are ornithopods. 4) A Miocene crocodylian specimen AMNH 7900 is referred to on line 117 as an indeterminate croc. But in figure 1 the same specimen is referred to as Alligator mississippiensis (and the caption does not make clear that this is a Miocene specimen).

5) Capitalise Cerapoda on line 198
6) In Figure 3, the grey clades are missing data, but on the figure itself these are referred to as "absence of uncinate". These are quite different things. Change the figure wording to reflect that this is missing data not genuine absence.
Reviewer #3 (Remarks to the Author): a really interesting paper focussing on an important topic -comments and suggestions below: line 44: are there any empirical data that support the mechanical hypothesis as both of these are text book references -do the authors think they should present this as a viable alternate hypothesis?
line 73 -could a reference to the presence of cartilaginous uncinate in crocodilians be added to flesh out this statement line 215 -wouldn't this suggest that these were present in the ancestor of both crocodilians and birds ?
In this document, line numbers in purple refer to the lines of the manuscript that the reviewers commented on. Line numbers in the revised change section refer to the lines of the revised manuscript submitted in this package.

Reviewer 1 (Dr Michael Pittman)
1. Line 29: The fossil sample dataset has a good number of fossil taxa but does not include pterosaurs to represent non-dinosaurian ornithodirans and has two taxa to cover noncrocodylian pseudosuchians (a notosuchian and phytosaurs by my count). The dataset is best suited to commenting on the Dinosauria ancestral condition and an edited paper with this narrowed focus would fit the requirements of this journal. As the manuscript, I believe noncrocodylian pseudosuchians from the major subclades need to be sampled to more robustly reconstruct the archosaur ancestral condition.

Revised changes:
Araripesuchus and three non-crocodylian pseudosuchians (NMMNH P50048, P60401, and YPM 6649) are the four non-crocodylian pseudosuchian taxa with uncinate scars sampled in this study. Sentences in the section "Ancestral state reconstruction for uncinate processes in archosaurs" and "Potential homology of uncinate processes across Archosauria" have been modified, so that the number of non-crocodylian pseudosuchians are explicitly mentioned.
The results at the node Archosauria have been retained because they suggest the possibility that uncinate processes are homologous across Archosauria. Although this result is tentative, including it in the paper indicates to the reader where our knowledge of the origin of uncinate processes stands at present, and will hopefully encourage further sampling of uncinate scars in fossil pseudosuchians.
However, sentences are added to line 197, to acknowledge that additional sampling from fossil pseudosuchians would be warranted in order to bolster the current result for Archosauria. The discussion in the section "Potential homology of uncinate processes across Archosauria" is modified in tone, to reflect the fact that few non-crocodylian pseudosuchians were sampled in this study.
2. Line 35: Any supporting references you could add?
Revised changes: Line 34. Reference has been added for the absence of uncinate processes in anhimid and megapodid birds, and for the fact that avian uncinate processes are normally ossified.
Line 41. Reference has been added for the fusion of uncinate processes to the vertebral ribs in birds. The presence of uncinate processes on the posteriormost cervical ribs, and the fusion between uncinate processes and their corresponding vertebral ribs observed in the avian skeletal materials housed at the University of Alberta Museum of Zoology (e.g. Falco sparverius UAMZ 4022) has been retained to support the referred literature.
3. Line 42: Any supporting references you could add?
Revised changes: Line 41. Osteology of the Reptiles by Romer (1956) has been added as a supporting reference.
4. Line 44: It would be helpful to explicitly state that the hypotheses are not mutually exclusive to help the non-specialist reader.
Revised changes: Line 42. sentence has been changed to "Two main hypotheses for the function of avian uncinate processes, which are not mutually exclusive have been proposed: mechanical reinforcement of the ribcage and ventilation".
5. Line 66: Recommend to use 'saurians' consistently, e.g., stegosaurians. Or you could just replace the use of 'saurians' in some places to 'saurs' Revised changes: '-saurian(s)' has been changed to '-saur(s)' throughout the text, except where a taxonomic name is being used as a modifier (lines 43, 64, and 125). Shartegosuchidae has been added to indicate that not all pseudosuchian lineages have been sampled in this study.
7. Line 84: There are only a few so I recommend to just say 'select notosuchians and ornithischians'.
Revised changes: Line 67. The sentence has been changed to "However, the selected notosuchian and ornithischian examples listed above notwithstanding, preserved uncinate processes are rarely found outside Pennaraptora.". 8. Line 136: Recommend to include clades before the genera as some readers won't be familiar with theropod phylogeny e.g., the dromaeosaurid Saurornitholestes langstoni (TMP 88.121.39). 9. Line 150: Recommend to include clades before the genera as some readers won't be familiar with ornithischian phylogeny e.g., in the hadrosaurs Gryposaurus (AMNH 5350, 5456) and Bactrosaurus johnsoni (AMNH 6553), … 10. Line 162: Recommend to include clades before the genera as some readers won't be familiar with ornithischian and sauropod phylogeny e.g., diplodocid sauropod Apatosaurus 11. Line 441: Adding clade names will be very helpful for reader that are not familiar with bird or crocodilian taxonomy. 12. Line 453: Same comment about clade names to help non-specialist readers.
Revised changes: Clade names have been added before the genera throughout the main text.
13. Line 165: This additional non-crocodylian pseudosuchian data point should be added to the main tree figure to better convey your taxon sampling.
14. Line 201: You could mention your notosuchian datapoint here. It seems it and the phytosaur (if it is an archosaur) are the only non-crocodylian pseuodosuchian taxa in your dataset? Revised changes: Line 235. Some text has been added to suggest that the advent of uncinate processes may have provided some increased structural stability to the trunk, although whether uncinate processes act this way even in modern birds still awaits rigorous analysis. We also have added sentences to mention that the uncinate processes would have provided additional surface area for ventilatory muscle attachment.
17. Line 210: An additional figure showing this across the tree would be very helpful to articulate where we are at now.
Revised changes: Line 551. A panel has been added to illustrate stages of the hypothetical evolutionary scenario described in the paper. We have chosen to add a panel instead an additional figure, because the hypothetical evolutionary scenario is probably clearer if we position the scenario next to the cladogram with results of ancestral state reconstruction.
18. Line 235: Can you be more explicit about why this is?
Revised changes: Line 258. The sentence has been changed to "An increase in metabolism in maniraptorans may be related to the origin of powered flight, which could not be explored further at present because the precise timings of an increased metabolism and the origin of avian powered flight remain uncertain", to clarify that additional data is warranted for further inference on the evolutionary relationship between the potential increased metabolism and the origin of powered flight.
Line 255. Sentence is added to describe the possibility that resting metabolic rates may be correlated to dimensions of cartilaginous uncinate processes. Accordingly, the resting metabolic rates may have increased before uncinate processes became ossified in pennaraptorans, if uncinate processes became elongated before the origin of pennaraptorans.
19. Line 469: Some of the insets are hard to make sense of. Recommend some further image postprocessing to improve contrast etc.
Revised changes: Lines 502 and 527. Font sizes and shape sizes have been adjusted for visibility. Photos have been sharpened to increase contrast where necessary, and colour of the rectangles have changed to orange for better visibility.
20. Line 481: I recommend to show results for all three mapping methods and at more internal nodes. There is a gap between the values visible at the three nodes and the colour coding of the clades that makes it difficult to evaluate the study results. Perhaps colour the branches so the reader can visually see the data within each colour coded clade? I feel that this figure should be self-contained without the need to refer to the supplement or raw data. The coloured circles at nodes and the rib cartoon were a bit confusing. Recommend to put the cartoon in the key or have the rib at the nodal symbol.
Recommend to make the square key symbol a circle too since the ML and MB values are also referring to nodal values.
Revised changes: Line 551. Figure 3 has been modified to include additional internal nodes for Saurischia and Theropoda ( Table 5 in the supplementary information is also updated), to demonstrate the results of ancestral state reconstruction leading to birds.
Internal nodes with values of ancestral state reconstruction are labelled by the initials of clades, to visually indicate their positions better visually on the cladogram. Branches with known uncinate processes or uncinate scars are colour coded and shaded based on the results using maximal parsimony on a time calibrated tree. Probability labels are changed to circles, and rib diagrams are removed.
Mapping all results together is great for comparisons. However, the results of three mapping methods are not identical for all nodes and branches, so mapping all three methods on top of each other would be confusing for readers.
1. Various taxonomic names are mentioned at various points without explaining them. For the benefit of the reader I would suggest "anhimid birds" rather than "anhimids", "neognath birds" instead of "neognaths" etc.
Revised changes: Explanatory terms (e.g. birds) have been added after the taxonomic names where appropriate.
2. At more than one point it is asserted that uncinate processes cannot be ancestral for saurians because they are absent in squamates (lizards and snakes). But if they are absent in squamates but present in rhynchocephalians, then the ancestral condition for Lepidosauromorpha is ambiguous, as is the ancestral condition for Sauria. You should tweak your text to reflect this ambiguity.
Revised changes: Line 39, 219. Sentences have been modified to reflect the ambiguity of the ancestral condition in both Lepidosauromorpha and Sauria.
Line 39. Sentence has been changed to "The rod-like cartilaginous uncinate processes of the rhynchocephalian Sphenodon punctatus may not be homologous to those of birds and crocodylians: most extant lepidosauromorphs lack uncinate processes.". Romer 1 has been added as a reference to support the statement that most extant lepidosauromorphs lack uncinate processes.
Line 219. Sentence has been changed to "… but the wider distribution of these structures across Sauropsida remains to be investigated".
3. On line 69 thescelosaurids and ornithopods are listed as if they are different taxa. But thescelosaurids are ornithopods.
Revised changes: Madzia, et al. 2 , one of the phylogenetic studies we consulted in constructing the tree used for the ancestral state reconstruction, recovered thescelosaurids as a group of basal neornithischians outside Ornithopoda. Similar results were obtained in several other studies of ornithischian systematics (e.g. Boyd 3 ). We therefore have continued to treat thescelosaurids as distinct from ornithopods.
We acknowledge, however, that some authors (e.g. Eberth,et al. 4 .) indeed continue to regard thescelosaurids as ornithopods, a view that was once widely held. 4. A Miocene crocodylian specimen AMNH 7900 is referred to on line 117 as an indeterminate croc. But in figure 1 the same specimen is referred to as Alligator mississippiensis (and the caption does not make clear that this is a Miocene specimen).